In skeletal muscle, excitation-contraction coupling involves a physical interaction between two different types of Ca2+ channels: the voltage-gated Ca2+ channels dihydropyridine receptors (DHPRs) located in the sarcolemma and the intracellular Ca2+ release channels of ryanodine receptor type 1 (RyR1) located on the sarcoplasmic reticulum (SR) membrane. In response to sarcolemmal depolarization, DHPRs undergo a conformational change that activates nearby RyR1. The subsequent massive release of SR Ca2+ into the myoplasm activates the contractile machinery. The DHPR-RyR1 mechanical interaction is bidirectional since RyR1 proteins dramatically enhance the ability of the DHPR to function efficiently as a Ca2+ conducting ion channel (Nakai et al., Nature 380:72-75 (1996); Dulhunty et al., Prog. Biophys. Mol. Biol. 79:45-75 (2000)).
Relaxation requires the ATP-dependent re-uptake of cytosolic Ca2+ into the SR or the removal of Ca2+ into the extracellular space by the sarcolemmal Ca2+ pump. Since the Ca2+ gradients across both cell and SR membranes are of the order of four magnitudes, cells expend a huge amount of energy to return elevated cytoplasmic Ca2+ to basal physiologic levels, i.e., to maintain Ca2+ control and homeostasis. This energetic effort can be revealed and quantitated by measuring the production of the ATP catabolites adenosine and inosine (Bünger, R. In: Topics and perspectives in adenosine research, pp. 223-235 (1987); Bünger, R. and S. Soboll, Eur J. Biochem 159: 203-13 (1986)), even when using a non-contractile cell model such as lymphocytes in an in vitro set-up.
Four clinically distinct hereditary human and muscle disorders are known to be associated with point mutations and deletions in the RyR1 gene: malignant hyperthermia (MH), central core disease (CCD), multiminicore disease (MmD), and nemaline rod myopathy (NM) (Jurkat-Rott, et al., J. Neural. 249:1493-1502 (2002); Taratuto A L. Curr Opin. Neural. 15:553-561 (2002)). In addition, heat, exercise, and stress induced muscle syndromes may be related to RyR1 defects, to some degree.
Malignant hyperthermia (MH) is a pharmacogenetic syndrome of skeletal muscle whose primary symptom is an abnormal response to volatile anesthetics and depolarizing skeletal muscle relaxants. Exposure to these agents during surgery can trigger uncontrolled Ca2+ release from the SR mainly through a mutated RyR1 gene leading to abnormal sensitivity of the RyR1 receptor. The increased intracellular Ca2+ load triggers a cascade of biochemical events that results in muscle rigidity, rhabdomyolysis, cardiac arrhythmia, acidosis, and eventually lethal hyperthermia. If not treated promptly by withdrawing the anesthetic and administration of dantrolene (an intracellular calcium blocker), the only drug available for MH episode treatment, mortality is extremely high (>80%). Initial MH symptoms can have a slow, clinically ambiguous onset, making an imminent MH episode difficult to recognize. Heat, stress, and exercise are other conditions that can induce MH-like symptoms.
Currently definitive MH diagnosis is made by means of the invasive, surgery-requiring caffeine halothane contracture test (CHCT) in North America, by a similar in vitro contracture test (IVCT) in Europe and by technically very difficult calcium induced calcium release (CICR) in Japan; all these tests use biopsied leg muscle (Vasitis lateralis). Other countries are using tests essentially similar to one or these tests. CHCT has a sensitivity of 97% (accurately detects MH susceptible individuals) with a specificity of only 78% (yields 22% false positives). However, because of the invasive surgery, the costs of the test, and the limited viability of the biopsied skeletal muscle samples, the CHCT must be completed within 5 hours post biopsy. Therefore, the muscle biopsies must be conducted at a certified MH diagnostic center or in close-by hospitals (currently there are only 6 MH diagnostic centers in North America). Because of these procedural and logistic complexities, it is estimated that only about 10% of all individuals eligible to undergo the CHCT test are actually tested (estimated from referrals to the North American Malignant Hyperthermia Hotline).
Central core disease (CCD) (also called Shy-Magee syndrome) is a hereditary myopathy characterized by proximal muscle weakness and skeletal deformities of the lower limbs (Taratuto A L. Curr Opin Neurol 15:553-561 (2002)). Diagnosis of CCD is established through histological identification of single, large amorphous areas of reduced oxidative enzyme activity in central or peripheral regions of Type I muscle fibers (i.e., central cores). However, in some CCD individuals, cores are not centrally located, but instead are found in peripheral regions of the muscle fiber. Furthermore, resting Ca2+ levels are apparently not elevated by CCD mutations that result in excitation-contraction uncoupling, indicating that an elevation in resting Ca2+ is not an absolute requirement for core formation (Avila et al., J. Gen. Physiol. 118:277-290 (2001); Avila et al., J. Gen. Physical. 121:277-286 (2003)).
Specific variants of multiminicore disease (MmD) (Ferreira et al., Ann. Neurol. 51:750-759 (2002); Jungbluth et al., Eurology 59:284-287 (2002)) and nemaline rod myopathy (NM) (Scacheri et al., Neurology 55:1689-1696 (2000)) have recently been shown to be also associated with mutations in the RyR1 gene. Skeletal muscle biopsies of patients suffering from MmD display multifocal, poorly circumscribed and short core-like lesions, whereas skeletal muscle obtained from individuals with NM exhibit rod-like structures when visualized using Gomori's trichrome stain in both Type I and II skeletal muscle fibers.
Significant clinical overlap exists between these related muscle disorders. For example, early-stage CCD may first present as a minicore myopathy (Ferreiro et al., Ann. Neurol. 51:750-75 (2002)), nemaline rods have been found adjacent to central cores in biopsies of some CCD patients (Scacheri et al., Neurology 55:1689-1696 (2000)), and CCD patients are often found to be MH susceptible (MHS), but MHS patients are unlikely to be CCD.
What is needed is a less complex, less time consuming, less invasive and more economical method for testing for RyR1 related diseases.